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Fluorescence and confocal microscopes operating principle. Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser scanning confocal microscopy (LSCM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation. [1]
In confocal laser-scanned microscopes, the full-width half-maximum (FWHM) of the point spread function is often used to avoid the difficulty of measuring the Airy disc. [1] This, combined with the rastered illumination pattern, results in better resolution, but it is still proportional to the Rayleigh-based formula given above.
Scanning laser ophthalmoscopy developed as a method to view a distinct layer of the living eye at the microscopic level. The use of confocal methods to diminish extra light by focusing detected light through a small pinhole made possible the imaging of individual layers of the retina with greater distinction than ever before. [4]
A 4Pi microscope is a laser scanning fluorescence microscope with an improved axial resolution. With it the typical range of the axial resolution of 500–700 nm can be improved to 100–150 nm, which corresponds to an almost spherical focal spot with 5–7 times less volume than that of standard confocal microscopy .
The three-dimensional point spread functions (a,c) and corresponding modulation transfer functions (b,d) of a wide-field microscope (a,b) and confocal microscope (c,d). In both cases the numerical aperture of the objective is 1.49 and the refractive index of the medium 1.52.
In particular, lateral resolution of 6 nm [4] and vertical resolution of 2–5 nm have been demonstrated. [5] [6] As in optical microscopy, the contrast mechanism can be easily adapted to study different properties, such as refractive index, chemical structure and local stress. Dynamic properties can also be studied at a sub-wavelength scale ...
Laser scanning is the controlled deflection of laser beams, visible or invisible. [1] Scanned laser beams are used in some 3-D printers, in rapid prototyping, in machines for material processing, in laser engraving machines, in ophthalmological laser systems for the treatment of presbyopia, in confocal microscopy, in laser printers, in laser shows, in Laser TV, and in barcode scanners.
Raman microscopy, and in particular confocal microscopy, can reach down to sub-micrometer lateral spatial resolution. [7] Because a Raman microscope is a diffraction-limited system, its spatial resolution depends on the wavelength of light and the numerical aperture of the focusing element. In confocal Raman microscopy, the diameter of the ...
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